Enzymes of the ubiquitin proteasome system (UPS) and their substrates are increasingly implicated in fundamental cellular processes, including cell proliferation, differentiation, transcriptional regulation, and stress response, amongst many others. With several hundred E3 ubiquitin ligases and ~50 E2 ubiquitin-conjugating enzymes, the UPS constitutes one of the most byzantine enzyme systems in eukaryotes. Whereas the basic principle of the E1-E2-E3 enzymatic cascade is now well established, the biological functions and regulation of ubiquitylation enzymes are largely a mystery and will remain so until all of their substrates have been revealed and functionally characterized. Thus, the identification of E2-E3 substrates has become the next frontier in the Ubiquitin Field, and the main goal of this grant application. The limited repertory of known substrates attests to the formidable challenges presented by the endeavor of matching enzymes with their substrates. Rather than relying on sporadic discovery, the project proposed here will build on a series of global approaches in S. pombe, which heavily employ cutting-edge proteomic technologies. These include whole proteome profiling by shotgun LC-MS/MS, and SPASS, a method for E2 substrate profiling by LC-MS/MS that was recently developed in the lab. In Aim 1, candidate substrates for the E2 Ubc7p, newly discovered by SPASS, will be validated using established genetic and biochemical techniques. In Aim 2, SPASS will be employed for systematic profiling of E3s that cooperate with three other fission yeast ubiquitin E2s, the essential Ubc4p and Ubc11p, as well as Ubc2p, an enzyme broadly implicated in DNA repair and chromatin structure. In Aim 3, comparative transcriptomic and proteomic profiling by shotgun LC-MS/MS will be used to identify novel candidate substrates of cullin-RING ubiquitin ligases. PUBLIC HEALTH RELEVANCE: The essential involvements of ubiquitylation enzymes (E2 and E3) in cellular regulation have placed them squarely at the center of prevalent human diseases, including cancer, viral infections, metabolic disease, and neurodegenerative disorders. Consequently, several pharmaceutical companies are already developing strategies for modulating UPS activity in a therapeutic context. A more detailed understanding of the exact pathways and substrates controlled by ubiquitylation enzymes such as enabled by the studies proposed in this application will be required to harness the full potential of such efforts.